Pancreatic cancer is one of the most lethal and devastating human malignancies, with a 5-year survival rate of approximately 6%. Early diagnosis of pancreatic cancer (CaP) is challenging due to lack of reliable diagnostic markers and limited understanding of the etiological factors. Heavy alcohol consumption constitutes a significant social problem and has been identified as a risk factor for pancreatic cancer. Alcohol use disorders frequently co-occur in military veterans and civilians; thus, these populations are vulnerable to face a disproportionate burden of increased risk to develop pancreatic cancer. This application will address evaluating the cellular and molecular mechanism by which alcohol increases pancreatic cancer risk. It will be highly significant and beneficial to understand the underlying mechanism of this complex disease association. Therefore, the overarching goal of this application is to examine the biological role of alcohol toxicity in the induction of pancreatic cancer and to identify genes mediating alcohol induced cellular transformation and progression of pancreatic cancer. Pancreatic cancer growth and persistence depend on the cancer stem cells (CSCs) that also induce recurrence and resist current chemo- and radiotherapies. Our preliminary data demonstrate that ethanol induced cellular transformation of normal pancreatic ductal epithelial cells. Chronic ethanol exposure induced malignant transformation of HPNE cells by causing inflammation (upregulated IL-6, IL-8, TNF?, and IL-1? genes, inhibited glutathione S-transferases (GSTs) and glutathione peroxidases (GPx), and induced stem cell markers). Interestingly, we identified that the expression pattern of Engrailed-2 (EN2), a homeobox-containing transcription factor was significantly altered in ethanol-transformed HPNE cells, and the induction of EN2 correlated with the rate of transformation. However, the biological function and molecular mechanism of EN2 in pancreatic cancer have never been examined. We demonstrate that overexpression of EN2 in human pancreatic normal ductal epithelial cells induces malignant transformation by generating CSCs, and regulating Notch-Hes1 pathway; suggesting that the expression of EN2 is oncogenic for pancreatic cancer. Further, we have shown that EN2 regulates transcription of pluripotency maintaining factors, that are required for sustaining the stem cells capacity for self-renewal and that the expression of EN2 is tightly regulated in cancer. EN2 is highly expressed in pancreatic CSCs and cancer cell lines, but not in normal pancreatic cells or normal pancreatic tissues. Further, we have shown that inhibition of EN2 attenuated CSC characteristics, Notch pathway, and tumor growth in mice. Based on our preliminary studies, we hypothesize that alcohol induces inflammatory environment during transformation resulting in induction of EN2, which acts as a key regulator of pluripotency and self-renewal and inhibition of EN2 in ethanol-transformed cells attenuates their tumorigenic potential. To examine the role of alcohol in pancreatic cancer and address the above hypothesis we propose to integrate functional, molecular, biochemical and histological approaches. Gain and loss of EN2 functional studies will be performed in vitro and in vivo. EN2 expression will be knocked down by CRISPR- Cas9 technology in vitro. Novel triple transgenic mice (LSL-KrasG12D; LSL-EN2+/+; Pdx-Cre) will be generated and effects of alcohol on pancreatic carcinogenesis will be compared in the presence or absence of EN2 (KrasG12D/EN2+/+, and KrasG12D/EN2-/- mice). We will examine the involvement of Notch-Hes1 pathway to assess the biological effects of alcohol mediated by EN2. The studies proposed in this application will enhance our understanding of the biology of ethanol-induced cellular transformation of normal pancreatic ductal epithelial cells, which will be significant for the management of pancreatic cancer.